1. Field of the Invention
This invention is concerned with a method of introducing endophytic fungi into grass. Herein, the endophytic fungi comprised of filamentous bacteria will be referred to hereafter as endophytes.
2. Description of the Related Art
Conventional methods of cultivating and growing grasses include the artificial crossing method, selection method, mutation method, cell fusion method and gene insertion method. Due to recent progress in biotechnology, the cultivation period which previously required 10 years or more, has been reduced to several years. As regards genetic insertion, several techniques exist such as a method using agrobacterium, the electroporation method and the particle gun method, and they are now being applied to a large variety of crops.
However, in the case of grasses which produce staple crops, it has been pointed out that this genetic insertion is extremely inefficient. It is dificult to infect grasses using the agrobacterium method, so genetic insertion is very difficult. As regards the electroporation method, a regeneration system has to be developed from the protoplast of the grass, and even if such a regeneration is possible, the characteristics of the plant may suffer damage due to growth mutations.
Concerning the particle gun method, since genes are introduced randomly into the plant organism or culture, the plant obtained frequently becomes a chimera.
In the case of grasses, cell culture techniques such as cell fusion or genetic insertion require complex operating procedures and as they are not very efficient, there are very few examples where they have successfully been applied efficiently and there are very few instances where they have been successfully developed on a practical level.
Bluegrasses known in the art include Kentucky bluegrass, Canada bluegrass, rough meadow grass, bulbous meadow grass, alpine meadow grass, wavy meadow grass, wood meadow grass, Balforth meadow grass, swamp meadow grass, broad leaf meadow grass, narrow leaf meadow grass, smooth meadow grass, spreading meadow grass and flattened meadow grass.
These plants are members of the group Pooideae, and also belong to the genus Poa which is a part of Poeae. Kentucky bluegrass, for example, is known by the name of Poa pratensis. Poa pratensis is a plant classified by Clayton et al (Genera Graminium, Grasses of the World, 1986).
Bluegrasses of the grass family, which comprises many types of grasses, are typified by Kentucky bluegrass. They are important to man, and are found in meadow and pasture.
Bluegrasses have a wide utility, and as they cover very large areas, they are subject to damage from disease or pests. Damage due to bluegrass worm (Japanese name Shibata moth) is severe, and in areas where pesticide sprays have not reached, grass can disappear overnight as soon as the larvae have hatched.
However, there are some wild plants in nature in which filamentous internal fungi, i.e. endophytes, live together with the plant. They grow particularly well in the gaps between cells, i.e. the intercellular spaces.
These endophytes, or symbiotic filamentous fungi, not only have no adverse effect on the host plant but in fact provide it with useful substances, and contribute to helping withstanding environmental stresses.
Enhancement by endophytes of plant properties is known from the literature, e.g. insect resistance (Siegel et al, 1987 Ann. Rev. Phytopathol. 25:293-315), disease resistance (Gwinn and Gavin, 1992, Plant Disease 76:911-914), environmental stress (drought, etc.) resistance (Arachevalta et al, 1989 Agron J. 81:83-90), and growth enhancement (Latch et al, 1985, N.Z.J. Agric. Res. 28:165-168). It is particularly well-known that, in perennial rye grass infected with endophytes, these endophytes improve insect resistance due to the repelling substances and alkaloids they produce.
Latch et al in New Zealand are searching for an endophyte known as an endosafe which has a low toxicity to livestock and excellent insect resistance by collecting and studying endophytes in perennial rye grass.
However, many of the plants in which these endophytes live have little utility, so it is necessary to introduce them into useful grasses. In this regard, attempts have already been made to introduce endophytes into perennial rye grass which is an important pasture grass. The techniques used may be broadly distinguished as artificial crossing and artificial inoculation.
In artificial crossing, useful characteristics are introduced by pollen using a plant infected by an endophyte as mer. However, in the conventional method, there were limitations on the species and strains which could be crossed with one another. In artificial inoculation, plants or culture tissues are inoculated with endophytes that have been isolated and cultivated.
The inoculation method is capable of introducing a wider range of types, but due to problems of technique regarding cultivation of endophytes, inoculation conditions and conditions of the plant itself, it is limited to perennial rye grass. To increase the infection rate, a method has been reported where callus is used as the plant tissue which is inoculated. However, according to this method, it is necessary to develop a plant regenerating system from the callus, hence the method was still limited to perennial rye grass.
The conventional cell cultivation method involves a troublesome procedure and requires considerable training. Its practical application was moreover difficult since culture mutations caused by transformation or cell fusion had an effect on the characteristics being introduced or on other traits.
In the genetic insertion method, it was not possible to introduce specific characteristics if it was not known which genes had an effect on the characteristics and type of plant.
Characteristics related to complex factors such as environmental stress could not be introduced by techniques such as genetic insertion. Moreover, plants grown by cell culture techniques were often found to exhibit decreased seed fertility. In the case of grasses this led to a decline of yield and was therefore fatal
In view of the above, growth techniques using endophytes or improvement of characteristics is a totally new approach to solve the above problems.
When endophytes are introduced into plants by artificial crossing, the type of plant is limited to those capable of being crossed with other types, hence such introduction is not possible within plants of the same type. Also undesirable forms of the endophyte primer (mer) are introduced into later generations, so this technique does not necessarily give useful plants.
Artificial inoculation on the other hand is limited to perennial rye grass due to problems in searching for endophytes and cultivation systems, and it has never been applied to other useful grasses. In callus inoculation, it is essential to develop a regenerating system of the plant into which the endophyte is to be introduced. Moreover, inoculation conditions had not been developed to increase the rate of infection.
At present, useful endophytes have been found only in perennial rye grass, tall fescue and meadow fescue which constitutes a major limitation to their introduction. In particular, these endophytes are foreign types, and no endophytes derived from foreign plants had yet been found that were adapted to Japanese environmental conditions.